Cyclins are essential activators of cyclin-dependent kinases (Cdks) and function to coordinate cell cycle transitions. Because Cdk activity is frequently rate-limiting for particular cell cycle transitions, it is imperative that Cdk activity be tightly controlled. G1 cyclins E and D1 are rate-limiting for cell cycle entry in mammals and inappropriately high levels of these cyclins have been linked to altered G1 control and to tumorigenesis. The levels of cyclins and their counterparts, Cdk inhibitors, are controlled both through transcriptional mechanisms and by ubiquitin-mediated proteolysis. In the previous funding cycle, we elucidated the SCF (Skpl, Cdc53, F-box protein) ubiquitin ligase pathway and found that it is responsible for regulated turnover of G1 cyclins in yeast, the Cdk inhibitor Sic1, and other regulatory proteins. In this renewal, we seek to further understand the SCF pathway and mammalian cyclin control through analyses of the F-box protein cyclin F, Fbw7, an F-box protein responsible for cyclin E turnover, and through identification of the cyclin D E3 ubiquitin ligase. Specific aim 1 will examine the role of cyclin F through analysis of mice and cells lacking the protein and through biochemical studies that address the mechanism of its turnover and its interaction with candidate substrates and Cdks. The role of Fbw7 in cell cycle control and development will be determined in Aims 2 and 3 through analysis of Fbw7-/- mice. Cells deficient in Fbw7 will be characterized for cell cycle defects and for stabilization of candidate target proteins. Mechanistic studies will establish whether the interaction of Fbw7 with cyclin E is spatially regulated. We will also explore the connections between Fbw7 and the Notch and Presenilin pathways through genetic and biochemical means. Aim 4 will use RNA interference in Drosophila and mammalian tissue culture cells, coupled with in vitro biochemical approaches, to search for the E3 responsible for cyclin D1 ubiquitination. Candidate E3s will be confirmed by in vitro reconstitution of cyclin D ubiquitination and their regulation explored.